The word TRAIL is known to technically define a molecule belonging to the family of the so-called “death ligands”, i.e. a family of Tumor Necrosis Factor molecules (TNF),
In practice, a TRAIL molecule can induce cell death in diseased tissues only, and spare healthy tissues, and for this characteristic it is a particularly interesting molecule for use in oncological treatments and in other biomedical fields.
TRAIL-based treatment of an organism affected by tumor cells may occur in two particular manners: in the former, the TRAIL molecule is chemically synthesized beforehand, in which case it is defined as recombinant TRAIL, and later administered to the tumor-affected organism; in the latter the TRAIL molecule may be introduced into a tumor-affected organism through a carrier consisting of a TRAIL-producing cell.
In the former case, the need was found for combined use of chemotherapeutic agents to enhance anti-tumor effects, because the latter have been found to progressively decrease, due to the very short half-life of the TRAIL molecule, i.e. of the order of 20/30 minutes, the high renal excretion rate of this molecule, and TRAIL resistance. The combination with chemotherapeutic drugs encourages the use of the recombinant TRAIL molecule due to its specific tumor cell eliminating capacity, but this combination is affected by high toxicity toward hepatocytes, lymphocytes and osteoblasts, and leads to undesired side effects for the organism.
Furthermore, due to short half-life and high excretion rate, repeated administration of chemotherapeutic drugs was required in combination with TRAIL and this caused a considerable increase of overall costs upon repeated TRAIL administrations. In the latter case, the TRAIL molecule is contained in a virus that is used as a vector therefor and allows release of its genetic makeup containing the TRAIL-encoding sequence, which is later translated into a protein and transferred to the membrane of the cell infected by such viral vector.
These infected cells become the medium that carries TRAIL in direct contact with target tumor cells, thereby causing apoptosis thereof.
The viral vectors used heretofore are vectors that belong to the lentivirus or adenovirus family.
The cells infected by these vectors can carry the TRAIL molecule to the location of the organism in which an anti-tumor effect, i.e. apoptosis of neoplastic cells is required.
For this purpose, multiple cell types are used, which all have a particular tropism for tumor disease sites: for example, hematopoietic stem cells and mesenchymal stem cells are used.
Nevertheless, this prior art still has certain drawbacks.
A first drawback is that the viral vector in use may have considerable restrictions of use due to its biological properties.
For instance, if an adenovirus is used, the greatest restriction consists in the inability of the viral genome to be stably integrated in the genome of infected cells, and this characteristic generates a transient, short-lasting form of TRAIL, which is designed to deplete, thereby causing a limitation of the duration of the therapeutic effect, like in the infusion of recombinant TRAIL.
An additional drawback, independent of the viral vector in use, is that the studies that have been conducted and published heretofore have concerned application of TRAIL only as a transmembrane protein and disregarded the existence of a biologically active domain of the molecule even in the form of a soluble ligand having a strong anti-tumor activity. This involved the generation of cell vectors that could only produce TRAIL cells as membrane proteins capable of inducing selective apoptosis of the target tumor cell only through direct contact allowed by interaction between the TRAIL on the carrier cell and its receptor on the target tumor cell; therefore, the membrane TRAIL carrying cell must be necessarily located in the proximity of or in contact with the tumor mass to ensure its therapeutic effect.
Another drawback is that the membrane TRAIL-carrying cell located in the tumor mass must survive and proliferate for the time required to exert its anti-tumor action; thus, the cell dose to be infused must be substantially comparable to the number of tumor cells to be eliminated, because cytotoxic action only occurs through cell contact.
As mentioned above, this causes high cell production costs and may give rise to side effects associated to infusion in the patient.
Another drawback is that the lack of an excreted TRAIL form has prevented more intensive pharmacokinetic studies on infused cells, and the duration of its effect with time could not be understood.
A further drawback is that carrier cells such as bone-marrow derived cells, have been used in the studies, without considering that these might produce molecules that can inhibit or even block the anti-tumor action of TRAIL molecules. The cell used as a carrier shall produce a small number of or no decoy receptors (such as OPGs), i.e. receptors capable of sequestering
Finally, the cell that is used as a carrier, in the case of stem cells, shall not have the TRAIL-specific receptors (DR4 and DR5) potentially capable of causing the suicide of the carrier cell, and hence hindering any therapeutic effect.
Recent studies have suggested that a tumor is a complex mixture composed by different cell types with different grade of malignancy (Mueller M M and Fusenig N E, Nature Reviews 2004). In several malignancies it has been identified a small population of tumor cells sharing many similarities with normal stem cells including self-renewal and multipotency, and for this reason defined cancer stem cells (CSC) (Visvader J E and Lindeman G J, Nature Reviews 2008).
CSC have the ability to regenerate a new CSC and to give rise to the variety of proliferating and differentiated cancer cells that make up of the bulk of a tumor (Al-Hajj M and Clarke M F, Oncogene 2004).
Inside the tumor CSC reside in a well-described compartment defined “CSC niche”.
The niche microenvironment regulates CSC stemness and proliferation influencing tumor progression and metastasis formation. In addition, it plays a protective role shielding them from environmental insults (Borovski T et al. Cancer Res, 2011).
Thanks to this defensive microenvironment in which CSC reside and due to specific cellular mechanisms that they develop including relative quiescence, high expression of several ABC drug transporters, active DNA-repair capacity and a resistance to apoptosis, CSC display an effective drug-resistance that allows them to survive to the chemotherapy treatment, repopulating the tumour and provoking disease relapse (Dean M et al. Nature Reviews 2005).
For this reason, CSC eradication represents the target for enduring curative effects after treatments. Unfortunately, these cells are resistant to the majority of the conventional therapies, therefore they have been very difficult to eliminate (Yi S Y and Hao Y B Cancer Treatment Reviews 2013).
In addition, due to their low frequency in tumor burden, the identification of CSC sub-population is not easy achievable. Many different markers have been used to characterize these cells. Among these, CD133 represents one of the most common used antigens for CSC identification and isolation (Grosse-Gehling P et al. J Pathol. 2013).
In addition, due to their stem-like properties these cells show high levels of stem-related genes such as OCT4, NANOG, SOX2 and c-Myc (Cabarcas S M et al. International Journal of Cancer 2011; Heddleston J M et al. Br J Cancer 2010). In particular, c-Myc is a well-known important regulator in the G1/S phase transition and self-renewal in stem cells and it has been extensively studied for its instrumental role in proliferation and growth of neoplastic cells (Shachaf C M and Felsher D W Cancer Res 2005). More, c-Myc has been recently recognized as an important regulator of stem cell biology connecting malignancy and “stemness”.
In CSC, c-Myc activates an embryonic stem cell-like transcriptional module, which strongly correlates with tumor metastasis, proliferation and maintenance (Jialiang Wang, et al. PlosOne 2008; Sheelu Varghese et al. PlosOne 2012; Robyn T. Sussman et al. Cancer Biology & Therapy 2007).
In these years in vitro and in vivo studies by applicants have demonstrated that the pro-apoptotic molecule TRAIL delivered by human pericytes extracted from adipose tissue (AD-PC) is particularly active to induce tumor cell apoptosis in several different epithelial and mesenchymal cancer types including cervical carcinoma, pancreas, colon, multiple myeloma, osteosarcoma, rhabdomyosarcoma and Ewing's sarcoma.
Interestingly, the anticancer effect displayed by TRAIL when delivered by AD-PC in some cases resulted even more effective than the one obtained with the recombinant protein (Grisendi G. et al Cancer Res 2010; Grisendi G. et al Stem Cells 2014).
The action of TRAIL is related to its interaction with functional TRAIL receptors (DR4 and DR5) that are widely expressed on cancer cells surface. Ligand and receptor binding trigger the apoptotic signal inside the target cell. This signal induces the activation of two apoptotic pathways, defined one as extrinsic and the other as intrinsic.
The extrinsic pathway is activated when TRAIL binds to DR4 and/or DR5, inducing receptor oligomerization and the recruitment of Fas-associated protein with death domain (FADD) on the intracellular side. This complex contributes to the formation of the DISC when inactive pro-caspase 8 is recruited.
Auto-activation of the DISC may promote the activation of caspase-8 causing the cleavage and subsequent activation of the effector caspases-3, -6, -7 leading to the execution of apoptosis. In addition to DNA fragmentation and membrane bebbling, the activated caspase-8 may promote the cleavage of a pro-apoptotic Bcl-2 family member called Bid.
Bid represents the connection with the intrinsic apoptotic pathway, whose activation concomitant with the extrinsic pathway may be necessary for an efficient apoptosis in certain cell types. Bid on turn interacts with Bax/Bak causing the release of the cytocrome c from mitochondria.
This contributes to the formation of the apoptosome with APAF-1 and pro-caspase-9 that gets activated the effector caspases, leading to the hallmarks of apoptosis (Johnstone R W et al. Nature Reviews 2008; Holoch P A European Journal of Pharmacology 2009).
It is known that c-Myc oncoprotein represents one of the major catalysts of TRAIL sensitivity and its overexpression dramatically sensitizes cells to the apoptotic action of TRAIL. One of the mechanisms by which c-Myc enforce TRAIL apoptotic signal is represented by up-regulation of DR4 and DR5 receptors (Sussman R T Cancer Biology & Therapy 2015; Wang Y et al. Cancer Cell 2004). Moreover c-Myc augments TRAIL-dependent activation of caspase-8 through transcriptional inhibition of FLIP, which antagonizes generation of active caspase-8 in DISC (Ricci M S et al. Molecular and Cellular Biology 2004).
Finally, it was shown that c-Myc controls a “feedback amplification loop” involving activation of Bak and amplifies the TRAIL-induced caspase-8-Bid signals to induce full-blown apoptosis (Nieminen A I et al. Cell Cycle 2007).